Genes and proteins, and their use

ABSTRACT

A protein from Group B  Steptococcus  is shown to be an outer surface protein and is a useful target for antimicrobial therapy.

REFERENCE TO RELATED APPLICATION

This Application is a continuation-in-part of U.S. Ser. No.09/868,352,filed on Jun. 15, 2001.

FIELD OF THE INVENTION

This invention relates to the identification of a bacterial gene andprotein, and its use. More particularly, it relates to its use intherapy, for immunisation and in screening for drugs.

BACKGROUND TO THE INVENTION

Group B Streptococcus (GBS), also known as Streptococcus agalactiae, isthe causative agent of various conditions. In particular, GBS causes:

Early Onset Neonatal Infection.

This infection usually begins in utero and causes severe septicaemia andpneumonia in infants, which is lethal if untreated and even withtreatment is associated with a 10-20% mortality rate.

Late Onset Neonatal Infection.

This infection occurs in the period shortly after birth until about 3months of age. It causes a septicaemia, which is complicated bymeningitis in 90% of cases. Other focal infections also occur includingosteomyelitis, septic arthritis, abscesses and endopthalmitis.

Adult Infections.

These appear to be increasingly common and occur most frequently inwomen who have just delivered a baby, the elderly and theimmunocompromised. They are characterised by septicaemia and focalinfections including osteomyelitis, septic arthritis, abscesses andendopthalmitis.

Urinary Tract Infections.

GBS is a cause of urinary tract infections and in pregnancy accounts forabout 10% of all infections.

Veterinary Infections.

GBS causes chronic mastitis in cows. This, in turn, leads to reducedmilk production and is therefore of considerable economic importance.

GBS infections can be treated with antibiotics. However, immunisation ispreferable. It is therefore desirable to develop an immunogen that couldbe used in a therapeutically-effective vaccine.

SUMMARY OF THE INVENTION

The present invention is based on the identification of a gene in GBS,and also related organisms, the product of which may be localised on theouter surface of the organism and therefore may be used as a target forimmuno-therapy.

According to one aspect of the invention, a peptide is encoded by a geneidentified herein as pho2-2, obtainable from Group B Streptococcus, or ahomologue or functional fragment thereof. Such a peptide is suitable fortherapeutic use, e.g. when isolated.

The term “functional fragment” is used herein to define a part of thegene or peptide which retains the activity of the whole gene or peptide.For example, a functional fragment of the peptide may be used as anantigenic determinant, useful in a vaccine or in the production ofantibodies.

A gene fragment may be used to encode the active peptide. Alternatively,the gene fragment may have utility in gene therapy, targetting thewild-type gene in vivo to exert a therapeutic effect.

A peptide according to the present invention may comprise the amino acidsequence identified herein as SEQ ID NO. 2, or a functional fragmentthereof.

Because of the extracellular or cell surface location, the peptide ofthe present invention may be a suitable candidate for the production oftherapeutically-effective vaccines against GBS. The term“therapeutically-effective” is intended to include the prophylacticeffect of vaccines. For example, a vaccine may comprise a peptideaccording to the invention, or the means for its expression, for thetreatment of infection. The vaccine may be administered to females priorto or during pregnancy to protect mother and neonate against infectionby GBS.

According to another aspect of the invention, the peptide or gene may beused for screening potential antimicrobial drugs or for the detection ofvirulence.

A further aspect of this invention is the use of any of the productsidentified herein, for the treatment or prevention of a conditionassociated with infection by a Group B Streptococcal strain.

Although the protein has been described for use in the treatment ofpatients, veterinary uses of the products of the invention are alsoconsidered to be within the scope of the present invention. Inparticular, the peptide or the vaccines may be used in the treatment ofchronic mastitis, especially in cows.

DESCRIPTION OF THE DRAWINGS

The invention is described with reference to the accompanying drawingwhere:

FIG. 1 is a graphic illustration of the average level of protectionoffered by anti-pho2-2 anti-sera against a challenge dose of GBS.

DESCRIPTION OF THE INVENTION

The present invention is described with reference to Group BStreptococcal strain M732. However, all the GBS strains and many otherbacterial strains are likely to include related peptides or proteinshaving amino acid sequence homology with the peptide of M732. Organismslikely to contain the peptide include, but are not limited to, S.pneumoniae, S. pyogenes, S. suis, S. milleri, Group C and Group GStreptococci and Enterococci. Vaccines to each of these may be developedin the same way as described for GBS.

Preferably, the peptides that may be useful for the production ofvaccines have greater than 40% sequence similarity with the peptideidentified herein. More preferably, the peptides have greater than 60%sequence similarity. Most preferably, the peptides have greater than 80%sequence similarity, e.g. 95% similarity. With regard to thepolynucleotide sequence identified herein, related polynucleotides thatmay be useful in the various aspects of the invention may have greaterthan 40% identity with the sequence identified herein. More preferably,the polynucleotide sequences have greater than 60% sequence identity.Most preferably, the polynucleotide sequences have greater than 80%sequence identity, e.g. 95% identity.

The terms “similarity” and “identity” are known in the art. The use ofthe term “identity” refers to a sequence comparison based on identicalmatches between correspondingly identical positions in the sequencesbeing compared. The term “similarity” refers to a comparison betweenamino acid sequences, and takes into account not only identical aminoacids in corresponding positions, but also functionally similar aminoacids in corresponding positions. Thus similarity between polypeptidesequences indicates functional similarity, in addition to sequencesimilarity.

Levels of identity between gene sequences and levels of identity orsimilarity between amino acid sequences can be calculated using knownmethods. In relation to the present invention, publicly availablecomputer based methods for determining identity and similarity includeone BLASTP, BLASTN and FASTA (Atschul et al., J. Molec. Biol., 1990;215:403-410), the BLASTX program available from NCBI, and the Gapprogram from Genetics Computer Group, Madison Wis. The levels ofsimilarity and identity provided herein, were obtained using the Gapprogram, with a Gap penalty of 12 and a Gap length penalty of 4 fordetermining the amino acid sequence comparisons, and a Gap penalty of 50and a Gap length penalty of 3 for the polynucleotide sequencecomparisons.

Having characterised a gene according to the invention, it is possibleto use the gene sequence to establish homologies in othermicroorganisms. In this way it is possible to determine whether othermicroorganisms have similar outer surface products. Sequence homologiesmay be established by searching in existing databases, e.g. EMBL orGenbank.

A fragment of the gene sequence disclosed herein may be used to preparea vaccine product, or as a probe in a diagnostic method, or to identifyhomologues in other microorganisms. Preferably the fragment will be atleast 20 nucleic acids, more preferably at least 30 nucleic acids, andmost preferably at least 70 nucleic acids.

When used to identify homologues, the gene sequence, or fragmentthereof, will preferably hybridise to the putative homologue understringent hybridisation conditions. Stringent hybridisation conditionsare those described by Sambrook et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press (1989), 1.101-1.104.According to this, hybridisation under stringent conditions means that apositive hybridisation signal is still observed after washing for 1 hourwith 1×SSC buffer and 0.1% SDS at 55° C., preferably at 62° C. and mostpreferably at 68° C.

Peptides or proteins according to the invention may be purified andisolated by methods known in the art. In particular, having identifiedthe gene sequence, it will be possible to use recombinant techniques toexpress the genes in a suitable host. Active fragments and homologuescan be identified and may be useful in therapy. For example, the peptideor its active fragments may be used as antigenic determinants in avaccine, to elicit an immune response. They may also be used in thepreparation of antibodies, for passive immunisation, or diagnosticapplications. Peptide fragments may be used to identify those parts ofthe protein that have the most favourable antigenic epitopes. Thefragments can be generated by methods known to those skilled in the art.For example, partial digests of the complete protein can be made andtested. Alternatively, synthetic peptide fragments can be made andtested. The fragments may be tested to establish which fragments elicitthe strongest immune response.

A therapeutic antibody of the invention will have affinity for theprotein (or peptide) of the invention and preferably should notcross-react with unrelated proteins or proteins in the patient.

Suitable antibodies which may be active against the peptide of theinvention, include monoclonal antibodies, or fragments thereof,including single chain fv fragments. Methods for the preparation ofantibodies will be apparent to those skilled in the art.

The preparation of vaccines is known to those skilled in the art.Vaccine compositions can be formulated with suitable carriers oradjuvants, e.g. alum, as necessary or desired, and used in therapy, toprovide effective immunisation against Group B Streptococci or othermicroorganisms that contain related proteins. The preparation of vaccineformulations will be apparent to the skilled person.

More generally, and as is well known to those skilled in the art, asuitable amount of an active component of the invention can be selected,for therapeutic use, as can suitable carriers or excipients, and routesof administration. These factors will be chosen or determined accordingto known criteria such as the nature/severity of the condition to betreated, the type or health of the subject etc.

The products of the invention may also be used in assays to screenpotential antimicrobial drugs. For example, the protein of the inventionmay be used as a target for antimicrobial therapy, to localise a drug tothe infecting microbe.

The products of the present invention were identified as follows:

A partial gene library of GBS (strain M732) chromosomal DNA was preparedusing the plasmid vectors pFW-phoA1, pFW-phoA2 and pFW-phoA3(Podbielski, A. et al. 1996. Gene 177:137-147). These plasmids possess aconstitutive spectinomycin adenyltransferase antibiotic resistancemarker, which confers a high level of spectinomycin resistance and istherefore easily selected. Furthermore, these vectors contain atruncated (leaderless) Escherichia coli phoA gene for alkalinephosphatase. The three vectors differ only with respect to the readingframe in which the leaderless phoA gene exists, as compared to anupstream in-frame BamHI restriction enzyme site. Because this truncatedE. coli phoA gene lacks the appropriate leader sequence for export ofthis enzyme across the bacterial membrane, extracellular alkalinephosphatase activity is absent when these plasmids are propagated in anE. coli phoA mutant (e.g. strain DH5α). The chromogenic alkalinephosphatase substrate, XP (5-bromo-4-chloro-3-indolyl-phosphate), doesnot enter intact bacterial cells and therefore only exported or surfaceassociated alkaline phosphatase activity can be detected. When exportedor surface associated alkaline phosphatase activity is present, thechromogenic XP substrate is cleaved to yield a blue pigment and thecorresponding bacterial colonies can be identified by their blue colour.

Plasmid DNA was digested to completion with BamHI and dephosphorylatedusing shrimp alkaline phosphatase. GBS genomic DNA was partiallydigested with Sau3AI, size fractionated on a sucrose gradient andfragments <1 kb in size were ligated into the prepared pFW-phoA vectors.E. coli strain DHS5α was chosen as the cloning host since it lacks afunctional phoA gene. Recombinant plasmids were selected on Luria agarcontaining 100 μg/ml of spectinomycin and 40 μg/ml of the chromogenic XPsubstrate. E. coli transformants harbouring plasmids containing GBSinsert DNA that complements the export signal sequence of the leaderlessphoA gene were identified by the blue colour of the colonies.Approximately 30000 different recombinant plasmids containing GBS insertDNA were screened in this manner and 83 recombinant plasmids, whichcomplemented the leaderless phoA, were chosen for further study.

From these experiments, several clones were selected each containing aplasmid containing a gene (or part thereof), which complemented theleaderless phoA.

A clone was selected containing a plasmid designated pho2-2. Thisplasmid contained a gene (or part thereof), which complemented theleaderless phoA. The nucleotide and deduced amino acid sequences of thegene are shown as SEQ ID NOS. 1 and 2, respectively.

A comparison of the amino acid sequence of pho2-2 was performed.

Homologues to the GBS pho2-2 gene product can be identified inEnterococcus faecalis, Escherichia coli (malK and afuC), Bacillussubtilis (glnO), Haemophilus influenzae (yebM and potA), Streptococcuspyogenes, Streptococcus pneumoniae and Salmonella typhimurium (malK).The E. faecalis, S. pyogenes and S. pneumoniae homologues wereidentified from genome sequence data and no annotations were availableas to the identity of the gene or gene products. In all other cases,homologues represented ATP-binding transport proteins that are part ofABC type transporters. Many of the components of ABC type transportersare membrane or cell surface associated, as these systems are involvedin the transport of macromolecules from the extracellular environment tothe intracellular compartment.

Having identified the gene in each clone it is then possible to obtainthe full-length gene sequence, as follows.

Using the identified and sequenced gene fragment, oligonucleotideprimers were designed for genomic DNA sequencing. These primers weredesigned so as to sequence in an ‘outward’ direction from the obtainedsequence. Once read, the sequence obtained was checked to see if the 5′and 3′ termini of the gene had been reached. The presence of thesefeatures was identified by checking against homologous sequences, andfor the 5′ end the presence of an AUG start codon (or acceptedequivalent) preceded by a Shine-Dalgarno consensus sequence, and for the3′ end, the presence of a translation termination (Stop) codon.

Upon identification of the full-length gene, primers were designed foramplification of full-length product. Primers used included restrictionenzyme recognition sites (NcoI at the 5′ end and EcoO109I at the 3′ end)to allow subsequent cloning of the product into the Lactococcalexpression system used.

PCR was carried out using the primers, and the products cloned into apCR 2.1 cloning vector (Invitrogen). Following confirmation of thepresence of the cloned fragment, the DNA was excised using therestriction enzymes NcoI and EcoO109I.

The vector into which this fragment was inserted was a modified versionof pNZ8048 (Kuipers, O. P. et al. (1998) J. Biotech 64: 15-21). Thisvector, harbouring a lactococcal origin of replication, achloramphenicol resistance marker, an inducible nisin promoter and amulticloning site was altered by the replacement of the multicloningsite with two 10× His tags, flanked on the 5-most end with an NcoI site,split in the middle with a multicloning site (including an EcoO109Isite), and a stop (termination) codon at the 3′ end of the His tags.

The gene of interest was inserted so that a 10× His tag was in the 3′position relative to the coding region. Following transformation of therecombinant plasmid into L. lactis (strain NZ9000-Kuipers, O. P. et al.(1998) supra), a 400 ml liquid culture was set up and translation of theprotein was induced by the addition of nisin to the culture. After a 2hour incubation, the cells were harvested and lysed by bead beating. Theresultant lysate was cleared by centrifugation, then passed over a metalaffinity (Talon, Clonetech) column. The column was washed repeatedlybefore bound proteins were eluted with Imidazole.

To identify fractions containing the His-tagged recombinant protein, analiquot from each fraction was analysed by SDS-PAGE, Western blotted andprobed with anti-His antibodies.

The recombinant protein obtained was then used to immunise New Zealandwhite rabbits, with pre-immune sera being harvested prior toimmunisation. Following a boost, the rabbits were sacrificed and seracollected. This sera was used in Western blots, ELISA and animalprotection models.

Using the sera obtained from the animal studies, immunosorption studieswere carried out.

Group B Streptococcus was grown in 20 ml Todd Hewitt broth (THB) for 8hours, harvested and resuspended in 5 ml PBS. 50 μl aliquots of thiswere used to coat wells in a 96 well plate (Nunc Immuno-Sorb). This wasleft at 4° C. overnight to allow for adsorbance of the bacteria onto theplate. Plates were washed twice with PBS, then blocked with 3% BSA inPBS for lhr at 37° C. Plates were again washed. Serial 10 fold dilutionsof the sera were made in PBS and 50 μl of these dilutions were added tothe wells of the plate, in duplicate. The plate was covered andincubated for 1 hr at 37° C. The plate was washed, then 50 μlanti-rabbit alkaline phosphatase conjugated secondary antibody at aconcentration of 1:5000 was added to each well. Following incubation at37° C. for an hour, the plate was washed again. 50 μl substrate (PNPP)was added to each well, and the reaction allowed to proceed for 30 minbefore the adsorbance was read at 405 nm.

The results showed that the antibody bound to the whole cells indicatingthat pho2-2 resides on the outer surface of the cell.

Animal protection studies were also carried out to test theeffectiveness of protection on the immunised rabbits.

Anti-sera against the pho2-2 outer surface protein and a further protein(pho3-9) identified in the same screen were raised in rabbits and theIgG from each anti-sera was purified using Protein A columnchromatography. Newborn pups from time-mated Sprague Dawley rats were‘immunised’ with 50 μl of the purified IgG intraperitoneally andreturned to different mothers for at least 5 hours before they werechallenged with the GBS.

The A909 strain of GBS was used as the challenge. The strain wasstreaked on to a blood agar plate, and allowed to grow over the weekendat room temperature. A single colony of GBS was used to produce theinoculum in THB. Following overnight growth, the GBS bacteria werecentrifuged, and an appropriate volume of PBS added to produce a finaldilution of 1×10⁶ CFU/ml GBS (5×10⁴ GBS/50 μl). The innoculum was kepton ice until use in the challenge assay in the rat pups.

The GBS was administered subcutaneously to each rat and the time ofchallenge recorded. All pups were monitored for approximately 63 hoursafter the GBS challenge.

Table A shows the percentage of pups that survived 63 hours afterchallenge with GBS. This data has been represented in a graph in FIG. 1.TABLE A Study date PBS Pho 2-2 Pho 3-9 Feb. 27, 2001 14 52 41 21 Mar.27, 2001 20 — 67 53 Apr. 3, 2001 17 — 46 — MEAN 19.50 51.50 37.00 SD5.57 11.27 22.63 SE 2.78 5.63 16.00

The pho2-2 protein offered significant protection against GBS infectioncompared to the PBS control and to the other outer surface proteinpho3-9.

1. A peptide encoded by a gene identified herein as pho2-2, obtainablefrom Group B Streptococcus, or a homologue thereof or a functionalfragment thereof.
 2. The peptide according to claim 1, having at least80% sequence identity to SEQ ID NO.
 2. 3. The peptide according to claim1, comprising the amino acid sequence identified herein as SEQ ID NO. 2.4. A polynucleotide encoding a peptide wherein said peptide is encodedby a gene identified herein as pho2-2, obtainable from Group BStreptococcus, or a homologue thereof or a functional fragment thereof.5. The polynucleotide, according to claim 4, wherein said polynucleotidehas 80% sequence identity with SEQ ID NO. 1, or a fragment thereof whichencodes a functional peptide.
 6. A host transformed to express a peptideencoded by a gene identified herein as pho2-2, obtainable from Group BStreptococcus, or a homologue thereof or a functional fragment thereof.7. The transformed host, according to claim 6, wherein said peptide hasat least 80% sequence identity to SEQ ID NO.
 2. 8. The transformed host,according to claim 6, wherein said peptide comprises the amino acidsequence identified herein as SEQ ID NO.
 2. 9. A vaccine comprising apeptide encoded by a gene identified herein as pho2-2, obtainable fromGroup B Streptococcus, or a homologue thereof or a functional fragmentthereof, or a means for expressing said peptide.
 10. The vaccine,according to claim 9, wherein said peptide has at least 80% sequenceidentity to SEQ ID NO.
 2. 11. The vaccine, according to claim 9, whereinsaid peptide comprises the amino acid sequence identified herein as SEQID NO.
 2. 12. The vaccine, according to claim 9, which comprises apolynucleotide encoding a peptide wherein said peptide is encoded by agene identified herein as pho2-2, obtainable from Group B Streptococcus,or a homologue thereof or a functional fragment thereof, wherein saidvaccine further comprises a means for expressing said peptide.
 13. Thevaccine, according to claim 12, wherein said polynucleotide has at least80% sequence identity with SEQ ID NO. 1, or a fragment thereof whichencodes a functional peptide.
 14. An antibody raised against a peptideencoded by a gene identified herein as pho2-2, obtainable from Group BStreptococcus, or a homologue thereof or a functional fragment thereof.15. A method for screening for potential pharmaceutical agents whereinsaid method comprises the use of a peptide encoded by a gene identifiedherein as pho2-2, obtainable from Group B Streptococcus, or a homologuethereof or a functional fragment thereof, to test for virulence.
 16. Themethod, according to claim 15, wherein said peptide has at least 80%sequence identity to SEQ ID NO.
 2. 17. The method, according to claim15, wherein said peptide comprises the amino acid sequence identifiedherein as SEQ ID NO.
 2. 18. A method for the treatment or prevention ofa condition associated with a bacterial infection wherein said methodcomprises administering an effective amount of a peptide encoded by agene identified herein as pho2-2, obtainable from Group B Streptococcus,or a homologue thereof or a functional fragment thereof.
 19. The methodaccording to claim 18, wherein the infection is a Group B Streptococcalinfection.
 20. The method according to claim 18, wherein the infectionis a focal infection.
 21. The method according to claim 18, wherein theinfection is a urinary tract infection.
 22. The method according toclaim 18, wherein said treatment is applied to an animal for veterinarytreatment.
 23. The method, according to claim 18, said peptide has atleast 80% sequence identity to SEQ ID NO.
 2. 24. The method, accordingto claim 18, wherein said peptide comprises the amino acid sequenceidentified herein as SEQ ID NO.
 2. 25. A pharmaceutical compositioncomprising a peptide encoded by a gene identified herein as pho2-2,obtainable from Group B Streptococcus, or a homologue thereof or afunctional fragment thereof, and a pharmaceutical carrier.
 26. Thepharmaceutical composition, according to claim 25, wherein said peptidehas at least 80% sequence identity to SEQ ID NO.
 2. 27. Thepharmaceutical composition, according to claim 25, wherein said peptidecomprises the amino acid sequence identified herein as SEQ ID NO. 2.